A Versatile Toolkit for Semi-Automated Production of Fluorescent Chemokines to Study CCR7 Expression and Functions

Chemokines guide leukocyte migration in different contexts, including homeostasis, immune surveillance and immunity. The chemokines CCL19 and CCL21 control lymphocyte and dendritic cell migration and homing to lymphoid organs. Thereby they orchestrate adaptive immunity in a chemokine receptor CCR7-dependent manner. Likewise, cancer cells that upregulate CCR7 expression are attracted by these chemokines and metastasize to lymphoid organs. In-depth investigation of CCR7 expression and chemokine-mediated signaling is pivotal to understand their role in health and disease. Appropriate fluorescent probes to track these events are increasingly in demand. Here, we present an approach to cost-effectively produce and fluorescently label CCL19 and CCL21 in a semi-automated process. We established a versatile protocol for the production of recombinant chemokines harboring a small C-terminal S6-tag for efficient and site-specific enzymatic labelling with an inorganic fluorescent dye of choice. We demonstrate that the fluorescently labeled chemokines CCL19-S6Dy649P1 and CCL21-S6Dy649P1 retain their full biological function as assessed by their abilities to mobilize intracellular calcium, to recruit β-arrestin to engaged receptors and to attract CCR7-expressing leukocytes. Moreover, we show that CCL19-S6Dy649P1 serves as powerful reagent to monitor CCR7 internalization by time-lapse confocal video microscopy and to stain CCR7-positive primary human and mouse T cell sub-populations.


Introduction
Chemokines, or chemotactic cytokines, are a family of small, secreted proteins that orchestrate cell migration [1,2]. Particularly, chemokines provide essential guidance cues for leukocytes and play a central role in the development and homeostasis of the immune system [3]. The human genome codes for at least 45 different chemokine proteins of 8-14 kDa. Chemokines are defined by their primary amino acid sequence and the presence of conserved cysteine residues, which form two structurally important disulfide bonds. They are grouped based on the position of the first two cysteines into CC (28 members), CXC (17 members), XC (2 members) and CX 3 C (1 member) chemokine ligands. Common structural features of chemokines further include a flexible, unstructured N-terminus, a central antiparallel three-stranded β-sheet and an overlaying C-terminal α-helix [1,2]. Functionally, they can be divided into inflammatory and homeostatic chemokines that ing from nature, we choose to covalently link a fluorophore to a recombinant chemokine in an enzymatic reaction. Bacillus subtilis expresses a specific Sfp synthase transferring 4 -phosphopantetheinyl groups from CoenzymeA (CoA) to a conserved serine residue of acyl or peptidyl carrier proteins (ACP or PCP), a process which can also be found in E. coli [36][37][38]. Such transferases are tolerant towards a wide range of molecules attached to CoA, including inorganic dyes [39]. Notably, ACP or PCP domains recognized by synthases can be minimized to an 11-12 amino acid short motif sufficient for enzymatic labelling [39,40]. The Sfp synthase was shown to have the highest affinity for a small, 12 amino acid sequence generated from recent YbbR-tags, referred to as the S6-tag consisting of the amino acid sequence GDSLSWLLRLLN [40]. Hence, we engineered the chemokines CCL19 and CCL21 as S6-tagged recombinant proteins, which can be purified on an ÄKTA automated FPLC and HPLC system, and that can easily be enzymatically labeled with a fluorophore of choice.

Recombinant Production, Automated Chromatography Purification and Enzymatic Fluorescent Labelling of CCL19 and CCL21
We have previously described a method to express and purify fluorescent CCL19 and CCL21 as fusion proteins with mRFP from the supernatant of transfected HEK293 cells, suitable to monitor chemokine receptor functions [33] and to identify stroma cell subsets in the thymus able to capture and present the chemokine [15]. A disadvantage of such an approach is that the fluorescent protein is more than twice the size of the chemokine itself and that it requests large quantities of transfected mammalian cells to produce the fluorescent chemokine fusion proteins. We therefore thought to establish an efficient and cost-effective method to produce and site-specifically label recombinant CCL19 and CCL21 in a semi-automated manner. Critically, random labelling or N-terminal modification of chemokines was not considered because such modifications are known to affect receptor binding and to frequently result in a loss of function [41,42]. To produce chemokines with a native N-terminus, we have adopted the protein His 6 -SUMO (also known as His 6 -SMT3) as a fusion protein (Figure 1a) to take advantage of the SUMO-specific protease Ulp1 (also known as SUMO protease) which cleaves the amide linking between the His 6 -SUMO-tag and the native chemokine [29,33,34,43]. At the chemokine's C-terminus we included a short flexible linker (single letter amino acid sequence SGGGGS) followed by the S6-tag (single letter amino acid sequence GDSLSWLLRLLN) for enzymatic fluorescent labelling using the Sfp synthase and an inorganic dye of choice [40] (Figure 1a).
The corresponding plasmids, pET-His 6 -SUMO-hCCL19-S6 and pET-His 6 -SUMO-hCCL21-S6, were expressed in the E. coli BL21(DE3) strain. The S6-tagged human chemokines can be purified from inclusion bodies and refolded following a detailed published protocol [34] and as described in the material and method section. We extended the standard chemokine purification and refolding procedure by introducing a 3-step purification process on an automated ÄKTA System, which allows easy manufacture upscaling associated with minimal manual process handling time (illustrated in Figure 1b). In a first step, bacterial inclusion bodies comprising the His 6 -SUMO-chemokine-S6 protein were dissolved and prepared for purification. In a second automated step, the sample was loaded on an immobilized metal (Ni 2+ ) affinity chromatography (IMAC) column to separate the tagged chemokine from residual bacterial proteins (Figure 2a,b). The His 6 -SUMO-chemokine-S6 protein was automatically eluted and slowly transferred to a new reaction tube comprising refolding buffer, facilitating the refolding of the chemokine by infinite dilution. After refolding, digestion buffer was applied containing the Ulp1 protease specifically cleaving off the His 6 -SUMO protein yielding in a chemokine-S6 protein possessing the native, mature chemokine's N-terminus (Figure 2a,b). The His 6 -SUMO protein was subsequently separated and removed from the chemokine by cation ion exchange chromatography (CIEX-C) within the ÄKTA automated purification process (Figure 2a,b). In a third polishing step, misfolded proteins were removed by reverse-phase HPLC, whereas the correctly folded chemokine was collected (Figure 2c,d), aliquoted and eventually lyophilized for storage. representation of the size matched expression constructs and the corresponding final flu labeled chemokine proteins. A cleavable His6-SUMO-tag is fused to the N-terminus of m human CCL19 and CCL21 followed by a short, flexible linker and the S6-tag for enzyma ling with the dye Dy 649P1 . Human CCL19-mRFP and CCL21-mRFP are included for size son. (b) Workflow overview of the chemokine production process. IMAC purification, c refolding, and CIEX purification can be performed in an automated fashion on a chroma system.
The corresponding plasmids, pET-His6-SUMO-hCCL19-S6 and pET-Hi hCCL21-S6, were expressed in the E. coli BL21(DE3) strain. The S6-tagged huma kines can be purified from inclusion bodies and refolded following a detailed protocol [34] and as described in the material and method section. We extended ard chemokine purification and refolding procedure by introducing a 3-step pu process on an automated ÄKTA System, which allows easy manufacture upsca ciated with minimal manual process handling time (illustrated in Figure 1b). In a bacterial inclusion bodies comprising the His6-SUMO-chemokine-S6 protein solved and prepared for purification. In a second automated step, the sample w on an immobilized metal (Ni 2+ ) affinity chromatography (IMAC) column to sep tagged chemokine from residual bacterial proteins (Figure 2a,b). The His6-SUM kine-S6 protein was automatically eluted and slowly transferred to a new rea comprising refolding buffer, facilitating the refolding of the chemokine by inf tion. After refolding, digestion buffer was applied containing the Ulp1 protea cally cleaving off the His6-SUMO protein yielding in a chemokine-S6 protein p the native, mature chemokine's N-terminus (Figure 2a,b). The His6-SUMO pr A cleavable His 6 -SUMO-tag is fused to the N-terminus of mature human CCL19 and CCL21 followed by a short, flexible linker and the S6-tag for enzymatic labelling with the dye Dy 649P1 . Human CCL19-mRFP and CCL21-mRFP are included for size comparison. (b) Workflow overview of the chemokine production process. IMAC purification, chemokine refolding, and CIEX purification can be performed in an automated fashion on a chromatography system.
Freshly prepared or reconstituted, recombinant human CCL19-S6 and CCL21-S6 were site-specific fluorescently labeled using the phosphopantetheinyl-transferase Sfp, which transfers the fluorescent dye (here Dy 649P1 ) conjugated phosphopantetheine moiety of CoA to the first serine residue of the S6-tag. An additional reverse-phase HPLC purification step served to separate the fluorescently labeled CCL19-S6 649P1 or CCL21-S6 649P1 , respectively, from residual substrate bound to the Sfp transferase (marked with asterisks in Figure 2c,d). Coomassie-stained SDS-PAGE gels with samples derived from the bacterial cell lysate before loading on the IMAC column, the IMAC flow through (FT), the IMAC eluate, the CIEX-C load after cleaving the His6-SUMO-tag, the CIEX-C flow through (FT) and CIEX-C eluate for the CCL19-S6 (a) and CCL21-S6 (b) purification steps illustrated in Figure 1b. (c,d) Representative HPLC chromatograms displaying the volume of gradient elution of CCL19-S6 (c) or CCL21-S6 (d) from a C-18 column used to separate correctly folded from misfolded chemokines (left panels) and to separate the fluorescently labeled chemokine from unlabeled chemokine, free substrate and the Sfp-CoA-Dy 649P1 (marked with *). Protein concentration was determined by measuring its absorbance at 280 nm (OD280); fluorescence was measured at 652 nm (OD652) and illustrated in red. (e,f). Representative Coomassie-stained SDS-PAGE gels (upper panels) and corresponding Western blots (WB, lower panels) of the same samples of native, S6-tagged and S6-tagged, as well as fluorescently labeled CCL19 (e) and CCL21 (f).
Freshly prepared or reconstituted, recombinant human CCL19-S6 and CCL21-S6 were site-specific fluorescently labeled using the phosphopantetheinyl-transferase Sfp, which transfers the fluorescent dye (here Dy 649P1 ) conjugated phosphopantetheine moiety of CoA to the first serine residue of the S6-tag. An additional reverse-phase HPLC purification step served to separate the fluorescently labeled CCL19-S6 649P1 or CCL21-S6 649P1 , respectively, from residual substrate bound to the Sfp transferase (marked with asterisks in Figure 2c,d). Figure 2c,d illustrate the high efficiency of chemokine labeling using this strategy. SDS-PAGE followed by Coomassie-staining or Western blotting revealed the correct size of the recombinant chemokine variants (Figure 2e,f) that match the calculated Figure 2. Purification and fluorescent labelling of human CCL19 and CCL21. (a,b) Representative Coomassie-stained SDS-PAGE gels with samples derived from the bacterial cell lysate before loading on the IMAC column, the IMAC flow through (FT), the IMAC eluate, the CIEX-C load after cleaving the His 6 -SUMO-tag, the CIEX-C flow through (FT) and CIEX-C eluate for the CCL19-S6 (a) and CCL21-S6 (b) purification steps illustrated in Figure 1b. (c,d) Representative HPLC chromatograms displaying the volume of gradient elution of CCL19-S6 (c) or CCL21-S6 (d) from a C-18 column used to separate correctly folded from misfolded chemokines (left panels) and to separate the fluorescently labeled chemokine from unlabeled chemokine, free substrate and the Sfp-CoA-Dy 649P1 (marked with *). Protein concentration was determined by measuring its absorbance at 280 nm (OD280); fluorescence was measured at 652 nm (OD652) and illustrated in red.
(e,f) Representative Coomassie-stained SDS-PAGE gels (upper panels) and corresponding Western blots (WB, lower panels) of the same samples of native, S6-tagged and S6-tagged, as well as fluorescently labeled CCL19 (e) and CCL21 (f).
Collectively, these data provide clear evidence that the new approach of generating fluorescently labeled chemokines results in biologically active CCL19-S6 649P1 and CCL21-S6 649P1 that efficiently elicit early CCR7 signaling pathways.

CCL19-S6 649P1 Specifically Binds to and Interacts with CCR7 on Transfected Cells
To complement our functional activity results, we tested the binding abilities of the fluorescently labeled chemokines to CCR7-expressing cells. For this, we incubated HeLa cells transiently expressing CCR7-EGFP at various temperatures for different time periods with 25 nM of the fluorescently labeled chemokines. At 10 • C, CCL19-S6 649P1 specifically bound to CCR7 transfected, but not to mock transfected cells (Figure 4a). Shifting the temperature to 22 • C, which still prevents receptor internalization, profoundly enhanced the specific binding of CCL19-S6 649P1 to CCR7-expressing cells (Figure 4a). Cell associated, chemokine-derived fluorescence was highest upon incubation at 37 • C (Figure 4a), where CCL19-S6 649P1 binds to CCR7 and subsequently promotes its co-internalization with the receptor [44]. Notably, CCL21-S6 649P1 interacted with both CCR7-expressing and vector control transfected HeLa cells at all temperatures tested (Figure 4b). This is likely due to the increased binding ability of CCL21 to glycosaminoglycans and charged carbohydrates on the cell surface [5,6,8,45]. The enhanced GAG binding ability of CCL21 compared to CCL19 [46], suggests that CCL21-S6 649P1 may be useful for visualizing the haptotactic CCL21 gradients rather than for labelling CCR7-expressing cells. Importantly, time-series experiments revealed that specific binding of CCL19-S6 649P1 to CCR7-EGFP expressing HeLa cells, but not to vector control transfected cells, was easily recorded after 10 min and reached a plateau after 25 to 40 min of incubation at 22 • C ( Figure 4c). Hence, CCL19-S6 649P1 is an excellent fluorescent reagent to specifically mark CCR7-expressing cells.

CCL19-S6 649P1 Is Internalized by CCR7-Expressing Cells
To further explore additional applications for the fluorescent chemokines, we incubated HeLa cells transiently expressing CCR7-YPet with fluorescently labeled CCL19-S6 649P1 and followed receptor-mediated internalization of the chemokine by live-cell confocal video microscopy. We focused on CCL19 as only this ligand is efficiently internalized by CCR7 as reported previously [44,47]. Upon addition to the medium at 37 • C, CCL19-S6 649P1 diffused within the medium and bound to CCR7-YPet expressed on the surface of transiently transfected HeLa cells. Subsequently, CCL19-S6 649P1 was rapidly internalized by CCR7-YPet expressing cells where the chemokine co-localized with its receptor in intracellular vesicles ( Figure 5, Supplementary Video 1). Critically, CCL19-S6 649P1 did not bind to and was not internalized by neighboring, untransfected HeLa cells ( Figure 5).

CCL19-S6 649P1 is Internalized by CCR7-Expressing Cells
To further explore additional applications for the fluorescent chemokines, we incubated HeLa cells transiently expressing CCR7-YPet with fluorescently labeled CCL19-S6 649P1 and followed receptor-mediated internalization of the chemokine by live-cell confocal video microscopy. We focused on CCL19 as only this ligand is efficiently internalized by CCR7 as reported previously [44,47]. Upon addition to the medium at 37 °C, CCL19-S6 649P1 diffused within the medium and bound to CCR7-YPet expressed on the surface of transiently transfected HeLa cells. Subsequently, CCL19-S6 649P1 was rapidly internalized by CCR7-YPet expressing cells where the chemokine co-localized with its receptor in intracellular vesicles ( Figure 5, Supplementary Video 1). Critically, CCL19-S6 649P1 did not bind to and was not internalized by neighboring, untransfected HeLa cells ( Figure 5).

CCL19-S6 649P1 is a Versatile Tool to Stain CCR7-Expressing Primary Lymphocyte Subsets
To further explore CCL19-S6 649P1 as tool to study endogenous CCR7 expression by primary lymphocytes, we isolated human peripheral blood mononuclear cells (PBMCs) from healthy donors and subsequently purified CD3 + T cells by MACS-sorting (Supplementary Figure S1a). We followed the optimized staining procedure established for CCR7 transfected cells to stain T cell sub-populations with our fluorescently labeled CCL19. We co-stained CD3-sorted T cells with CCL19-S6 649P1 together with fluorescently labeled antibodies for CD4 (marking T helper cells), CD45RA (naïve T cells), CD45RO (memory T cells) or CCR7 (Figure 6a,b). Importantly, CCL19-S6 649P1 marked the same T cell sub-populations as two distinct commercially available anti-CCR7 antibodies (Figure 6a). In fact, CCL19-S6 649P1 marked about 40% of CD4 + , ~40% of CD45RA + , and ~20% of CD45RO + T cells derived from human peripheral blood. Notably, CCL19-S6 649P1 binding does not interfere with the CCR7 antibody staining, and hence does not compete for the same binding site on the receptor (Supplementary Figure S1b). Finally, we also used our CCL19-S6 649P1 to stain mouse CD3 + T cells isolated from the spleen. Indeed, CCL19-S6 649P1 readily marked CCR7-expressing mouse T cells (Figure 6c).

CCL19-S6 649P1 Is a Versatile Tool to Stain CCR7-Expressing Primary Lymphocyte Subsets
To further explore CCL19-S6 649P1 as tool to study endogenous CCR7 expression by primary lymphocytes, we isolated human peripheral blood mononuclear cells (PBMCs) from healthy donors and subsequently purified CD3 + T cells by MACS-sorting (Supplementary Figure S1a). We followed the optimized staining procedure established for CCR7 transfected cells to stain T cell sub-populations with our fluorescently labeled CCL19. We co-stained CD3-sorted T cells with CCL19-S6 649P1 together with fluorescently labeled antibodies for CD4 (marking T helper cells), CD45RA (naïve T cells), CD45RO (memory T cells) or CCR7 (Figure 6a,b). Importantly, CCL19-S6 649P1 marked the same T cell sub-populations as two distinct commercially available anti-CCR7 antibodies (Figure 6a). In fact, CCL19-S6 649P1 marked about 40% of CD4 + ,~40% of CD45RA + , and~20% of CD45RO + T cells derived from human peripheral blood. Notably, CCL19-S6 649P1 binding does not interfere with the CCR7 antibody staining, and hence does not compete for the same binding site on the receptor (Supplementary Figure S1b). Finally, we also used our CCL19-S6 649P1 to stain mouse CD3 + T cells isolated from the spleen. Indeed, CCL19-S6 649P1 readily marked CCR7-expressing mouse T cells (Figure 6c). In summary, we herein describe a method for the production, ÄKTA automated purification and fluorescent labelling of functional human CCL19-S6 649P1 and CCL21-S6 649P1 . Moreover, we demonstrate that CCL19-S6 649P1 serves as versatile tool to study CCR7 function and expression of primary human and mouse leukocyte sub-populations.

Discussion
In the present study, we have established a method for the cost-effective production, ÄKTA-automated FPLC and HPLC purification and fluorescent labelling of chemokines using the important lymph node homing chemokines CCL19 and CCL21 as a proof-ofconcept. However, this approach is generally applicable to any other chemokine. To achieve this, we expressed the chemokines in a bacterial host system, which can easily be adapted for large-scale protein production. Importantly, chemokines are secreted proteins possessing a mature N-terminus that results from the removal of the signal peptide. The mature N-terminus is critical for its biological activity [41]. Hence, care must be taken In summary, we herein describe a method for the production, ÄKTA automated purification and fluorescent labelling of functional human CCL19-S6 649P1 and CCL21-S6 649P1 . Moreover, we demonstrate that CCL19-S6 649P1 serves as versatile tool to study CCR7 function and expression of primary human and mouse leukocyte sub-populations.

Discussion
In the present study, we have established a method for the cost-effective production, ÄKTA-automated FPLC and HPLC purification and fluorescent labelling of chemokines using the important lymph node homing chemokines CCL19 and CCL21 as a proof-ofconcept. However, this approach is generally applicable to any other chemokine. To achieve this, we expressed the chemokines in a bacterial host system, which can easily be adapted for large-scale protein production. Importantly, chemokines are secreted proteins possessing a mature N-terminus that results from the removal of the signal peptide. The mature N-terminus is critical for its biological activity [41]. Hence, care must be taken when chemokines are produced as recombinant proteins because bacteria lack corresponding signal peptidases to properly process the chemokine's signal peptide. Expressing chemokines without a signal peptide in bacteria is principally possible but introduces an N-terminal methionine to the mature protein, which often hampers chemokine functions. To circumvent these caveats, we fused a His 6 -SUMO-tag to the mature N-terminus of the chemokine. This facilitates on the one hand the efficient and straightforward affinity purification of the recombinant protein from bacterial inclusion bodies on Ni 2+ columns. On the other hand, the His 6 -SUMO-tag can easily be removed by the SUMO-specific protease Ulp1, as Ulp1 recognizes the SUMO fold and specifically cleaves the amide bond between the His 6 -SUMO-tag's C-terminus and the native, mature chemokine N-terminus [34,48]. For effective fluorescent labelling of the chemokine, we C-terminally introduced a short, six amino acid long flexible linker followed by a twelve amino acid long S6-tag. The S6-tag can be enzymatically and site-specifically labeled with the Sfp synthase [40] using fluorophore-conjugated CoA as substrate [39]. Most recombinant chemokines expressed in bacteria normally accumulate in high amounts in inclusion bodies and are unfolded. However, unfolded chemokines can be refolded by dialysis or infinite dilution [41,48]. An elegant approach is the on-column refolding of recombinant chemokines [49]. We further developed this approach to purify and refold the chemokines CCL19-S6 and CCL21-S6 on an automated ÄKTA chromatography system (Figure 1b). Our approach allows to cost-effectively produce decent amounts of fluorescent chemokines.
In general, various forms of commercially available or self-made fluorescent chemokines are sparsely available but have successfully been used to detect atypical and classical chemokine receptors on cell subsets, to sort chemokine receptor-positive cells, to study chemokine uptake or to investigate cell migration. For instance, CCL2-mCherry was used to identify the scavenging activity of CCR2 during monocyte migration [50]. An AlexaFluor-647-labeled CCL22 served to detect and characterize stromal cell populations expressing the atypical chemokine receptor ACKR2 upon intratracheal or intravenous application of the chemokine in mice [32]. A fluorescently labeled chimeric CXCL11_12 chemokine was exploited as unique tool to measure the scavenging activity of the atypical chemokine receptor ACKR3 by marginal zone B cells in vitro and in vivo [51,52]. Human CCL20s Dy649P1 and mouse CCL20y AF647 served to identify ACKR4 as the scavenger receptor for this chemokine in vitro and in vivo [43]. A site-specific fluorescently labeled CCL21 was used to investigate the heparinase-sensitive binding of the chemokine to CCR7 in receptor transfected HEK293 cells [35]. We have successfully used CCL19-mRFP and CCL21-mRFP to monitor CCR7 and ACKR4 functions in vitro [33]. In the present study, we used our newly produced and fluorescently labeled CCL19-S6 649P1 to investigate early CCR7 signaling events and to monitor endocytosis of the chemokine/receptor complex. Moreover, we exploited CCL19-S6 649P1 to label CCR7-expressing human and mouse T cell sub-populations in leucocyte cell suspensions. These fluorescently labeled chemokines seem also ideal tools to identify ACKR4-expressing cells and to study ACKR4 functions, such as chemokine scavenging, as well as chemokine gradient formation and maintenance. Overall, we present a cost-effective approach to produce decent amounts of fluorescently labeled chemokines as versatile tools to study and visualize chemokine receptor functions.

Plasmids
The cloning of the plasmids pET-His 6 -SUMO-hCCL19 and pET-His 6 -SUMO-hCCL21 used for recombinant production of native, mature human CCL19 and CCL21 have been described previously [29,53]. The coding sequence for His 6 -SUMO-tagged chemokines were amplified by PCR using the forward primer primers 5 -CCC TCT AGA AAT AAT TTT GTT TAA CTT TAA GAA GGA GAT ATA CAT ATGG and the reverse primer 5 -CAG GTG CTC GAG TTA TTA GTT CAG CAG GCG CAG CAG CCA GCT CAG GCT ATC GCC GCT GCC GCC GCC GCC GCT ACT GCT GCG GCG CTT CAT CTTGG for CCL19 and 5 -CAGGTGCTC GAG TTA TTA GTT CAG CAG GCG CAG CAG CCA GCT CAG GCT ATC GCC GCT GCC GCC GCC GCC GCT TGG CCC TTT AGG GGT CTG TG for CCL21 was used, which introduced a SGGGGS-peptide linker and the S6-peptide tag (GDSLSWLLRLLN) to the C-terminal end of the chemokine. The amplified PCR fragments were re-cloned into the XhoI and XbaI restriction sites of pET-His 6 -SUMO revealing pET-His 6 -SUMO-hCCL19-S6 and pET-His 6 -SUMO-hCCL21-S6, respectively.

SDS-PAGE and Western Blot Analysis
Purified recombinant proteins or bacterial lysate samples were precipitated using ethanol or were directly mixed with 5× gel sample buffer, respectively. Proteins were denatured at 95 • C for 5 min and separated by SDS-PAGE. SDS-gels were stained with Instant Blue (Expedeon, Abcam, Cambridge, MA, USA) or semi-dry blotted onto nitrocellulose membranes (Whatman, GE Healthcare, Chicago, IL, USA) for Western blotting. Membranes were incubated with primary antibodies overnight at 4 • C followed by incubation with the corresponding secondary antibody at room temperature for 2h. Blots were developed using chemiluminescence using the Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA) and analyzed on a ChemiDoc XRS Imaging System (Bio-Rad, Hercules, CA, USA).
Transient transfection of HeLa cells was performed using the Neon Transfection System (ThermoFisher, Waltham, MA, USA) according to the manufacturer's protocol and the 100 µl transfection kit. In brief, 5 × 10 5 cells were transfected with 10 µg of total plasmid DNA, pulsed twice for 35ms at 1.005V before seeding in pre-warmed DMEM supplemented with 20% FCS. Cells were used for subsequent experiments 36-48h post-transfection.

Isolation of Primary Human and Mouse Leukocytes
Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh whole blood samples of voluntary healthy donors using BD Vacutainer CPT with an integrated Ficoll gradient (BD Biosciences, San Jose, CA, USA) according to the manufacturer's protocol. Erythrocytes were removed by treating PBMCs with red blood cell lysis buffer (Biolegend, San Diego, CA, USA). Subsequently, CD3 + T cells were isolated using the Pan T cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and allowed to recover in RPMI-1640 medium (Pan-Biotech, Aidenbach, Switzerland) supplemented with 10% FCS (Lonza, Basel, Switzerland) overnight. Individual healthy donors gave written consent and blood donations were approved by the local ethics committee. Mouse splenocytes were isolated from the spleen of C57BL/6J mice kindly provided by the Animal Facility of the University of Konstanz. The organ collection was approved by the Review Board of Regierungspräsidium Freiburg in accordance with German Animal Protection Law (T-19/04TFA).

Chemokine-Mediated [Ca 2+ ] i -Mobilization
To investigate chemokine-mediated changes in concentrations of intracellular free calcium, 300-19 cells expressing CCR7 were washed with pre-warmed calcium flux buffer (145 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 1 mM sodium phosphate, 5 M HEPES, pH 7.5) and loaded with 4 mM Fluo-3 AM (ThermoFisher, Waltham, MA, USA) at 37 • C for 25 min. Cells were washed, adjusted to 3 × 10 6 cells/mL and Fluo-3 fluorescence was recorded over time on a LSRII flow cytometer (BD Biosciences, San Jose, CA, USA). Cells were stimulated after 30 sec with 50 nM chemokine as indicated. Towards the end of an experiment, cells were treated with 1 µM ionomycin to determine maximal release of [Ca 2+ ] i . Data were analyzed using the FACS Diva Software (BD Biosciences, San Jose, CA, USA).

Chemotaxis Assay
Two-dimensional cell migration assays were performed using 24-well Transwell plates (Corning, Corning, NY, USA) with 5 µm pore size. In brief, 1 × 10 5 cells were seeded into the upper compartment allowing cell migration towards different concentrations of chemokines in the lower compartment. After 3h, migrated cells were collected and the cell number was determined by flow cytometry. The number of migrated cells was corrected for randomly migrated cells towards medium without chemokines.

Chemokine Binding and Uptake Assay
HeLa cells were transiently transfected with CCR7-EGFP and seeded at a density of 1 × 10 5 cells in 12-well plates. After 36-48h, cells were washed with PBS and pre-incubated at 10 • C, 22 • C or 37 • C for 15 min to equilibrate. Subsequently, cells were incubated with 25 nM of fluorescently labeled chemokines for various time points. Chemokine binding (at 10 • C or 22 • C) and chemokine uptake (at 37 • C) of CCR7-EGFP positive cells was determined after extensive washing on a BD LSRII flow cytometer (BD Biosciences, San Jose, CA, USA).

Chemokine Receptor Surface Staining
Human peripheral blood-derived CD3 + T cells and mouse splenocytes were washed with calcium flux buffer. In total, 1.5 × 10 5 cells per assay point were incubated according to the manufacturer's protocol at room temperature for 20 min with the corresponding antibody or with 50 nM fluorescently labeled chemokine. Cells were washed with calcium flux buffer and analyzed on a BD LSRII flow cytometer (BD Biosciences, San Jose, CA, USA).

Confocal Live Cell Imaging
HeLa cells were transiently transfected with CCR7-YPet and seeded into 35 mm µ-Dishes (Ibidi, Fitchburg, WI, USA). Adherent cells were washed and equilibrated at 37 • C with HEPES-buffered DMEM without phenol red (Gibco, ThermoFisher, Carlsbad, CA, USA) on a temperature-controlled TCS SP5 II (Leica, Wetzlar, Germany) for 10 min. Cells were stimulated with 10 nM of Dy 649P1 fluorescently labeled CCL19-S6 and images were acquired using a 63× oil immersion objective. Acquired images were analyzed using FIJI [54].